In a virion (virus particle), the viral nucleic acid is covered by a protein capsid, and in some of the more complex animal viruses, the capsid itself is surrounded by an envelope containing membrane lipids and glycoproteins. Among these enveloped viruses, the ones receiving most attention today are the different retroviruses, especially HIV (Human immunodeficiency virus). The name retrovirus comes from the direction of the flow of genetic information for these viruses, from RNA to the DNA of the cell. Some retroviral strains are found to be symbiotic in one species and pathogenic in another. These findings indicate a potentially severe problem with xenotransplantation (transplantation of animal organs in humans). Both endogenous and exogenous retroviruses can contribute to vertical and horizontal transmission of genetic material within and between species and provide mechanisms for evolution of new pathogenic agents.
During the last decade there has been an increasing demand for methods for diagnosis and monitoring of retroviral infections as well as for research on the pathogenesis of new retroviruses. The state of the art is best illustrated by the methods currently used in the management of HIV infection. In the laboratory HIV-infection is detected and monitored by measurement of a) antibodies to HIV antigen, b) circulating HIV antigen, c) virus isolation, d) HIV RNA in circulation, and e) provirus DNA integrated in cells. The method of choice depends on the purpose of the test, and the stage of disease.
The currently accepted marker for viral load in the clinical setting is measurement of copy number of HIV RNA in plasma This can be accomplished either by PCR, NASBA, or by branched DNA techniques [Revets el al., 1996]. Currently used assays have a detection limit of 20 RNA copies/ml plasma [Perrin et al., 1998]. As all techniques for detection of viral nucleic acids are based on the binding of sequence specific primers, there is a significant risk that they will not hybridize to new strains or sequence variants in a sample.
Another important aspect concerning in vitro measurements of infection parameters emerges when extrapolating the results for the in vivo situation. Several of the laboratory techniques involve an amplification stage during which a selection step occurs. In the worst case the major viral variants amplified are those best matching the primers used, not the most abundant in the in vivo situation.
Direct measurement of viral enzymes in samples from the patient's blood would therefore be an attractive alternative to cell culture or the nucleic acid based methods both for quantification of viral replication and for studies of the effect of antiviral substances. The obstacles to overcome are both the assay sensitivity and to reproducibly separate the enzyme from host-specific enzymes and activity inhibiting substances.
The present invention enables measurement of an enzyme activity from an enveloped virus present in a biological sample by concentrating and recovering the enzyme activity without isolating the virions.
The prior art methods designed for concentration and purification of different viruses are either specific methods based on the antigenic properties of the virus proteins or generalized methods based on basic chemical or physical characteristics of the virus. Only the latter type of methods can be considered relevant for purification of antigenically highly variable retrovirus like HIV.
Viruses have traditionally been partly purified by different types of centrifugation, either by just pelleting the virus or in combination with the use of precipitating agents such as polyethylene glycol (U.S. Pat. No. 5,661,022). Centrifugation in density gradients results in more pure virus preparations (U.S. Pat. No. 4,729,955).
Another approach has been to bind the virus to different types of adsorbents. Porath and Jansson (U.S. Pat. No. 3,925,152) used insoluble organic macroporous polymers selected from the group consisting of agar, agarose, dextran and cellulose containing amphoteric substitutents composed of both basic nitrogen containing groups and carboxylate or sulphonated groups: Adsorbed viruses could in their system be eluted with solutions of successively different ionic strength to obtain virus variant separation. Their pioneering work has during the last two decades been followed in many related purification techniques based on interaction of viruses with gels with either anionic or cationic ionexchange residues (U.S. Pat. No. 3,655,509). Retroviruses and the related hepatitis B virus has for example been purified using both cation exchange (U.S. Pat. No. 5,447,859, U.S. Pat. No. 4,138,287) or anion exchange (U.S. Pat. No. 5,837,520, U.S. Pat. No. 5,661,023). The methods cited above are all based on binding the virus at low ionic strength and elution at high ionic strength.
Yet another approach for purification of virus and viral nucleic acid is based on binding virus to a water insoluble cross-linked polyhydroxy polycarboxylic acid polymer (U.S. Pat. No. 5,658,779). The virus is bound to the polymer at pH 6.0 to 8.0, probably by hydrophobic interactions and can be desorbed at pH 8.0 to 11.0. One interesting application of this technique concerns isolation of viral nucleic acid. In one such example bacteriophages from a bacterial lysate are in a first step adsorbed to the polymer. The polymer is then washed and the bound virus is disrupted using either a chelating agent, like EDTA, or a denaturating agent, like SDS. The viral proteins then adsorb to the polymer and a viral nucleic acid essentially free of interacting proteins is achieved. Use of a similar approach for purification of viral proteins is claimed but the technique described will release the viral proteins together with all other cellular and viral components adsorbed to the polymer.
The present inventors have conducted extensive research on measurements of the activity of the viral enzyme reverse transcriptase (RT). RT activity has previously been measured directly in serum or plasma [Awad, R. J. K. et al 1997, Ekstrand et al 1996]. One problem encountered in these early studies was the induction of high titers of RT activity inhibiting antibodies soon after primary infection. Another was the occasional occurrence of RT inhibitors in samples from HIV negative controls.